The ability to move DNA from one cell to another is a powerful tool in modern molecular biology and has profound practical implications for human health. Recombinant proteins produced by such manipulations are becoming widely accepted treatments for a number of human diseases and play major roles in agriculture. Though far less developed, the field of human gene therapy also has been and will continue to be influenced by improvements in technologies for manipulating DNA.
Gene therapy is a medical intervention in which a small number of the patient's cells are modified genetically to treat or cure any condition, regardless of etiology, that will be ameliorated by the long-term delivery of a therapeutic protein. Thus, almost all diseases that are currently treated by the administration of proteins, as well as several diseases for which no treatment is currently available, are candidates for treatment using gene therapy.
In somatic cell gene therapy, somatic cells (i.e., fibroblasts, hepatocytes, or endothelial cells) are removed form the patient, the cells are cultured in vitro, the gene(s) of therapeutic interest are added toe h the cells, and the genetically-engineered cells are characterized and reintroduced into the patient. The means by which these five steps are carried out are the distinguishing features of a given gene therapy system.
One example of a use of gene therapy is in treating hemophilia B, a bleeding disorder caused by a deficiency in Factor IX, a protein normally found in the blood. As a candidate for gene therapy cure, an afflicted patient would have an appropriate tissue removed (i.e., bone marrow biopsy to recover hematopoietic stem cells, phlebotomy to obtain peripheral leucocytes, a liver biopsy to obtain hepatocytes or a punch biopsy to obtain fibroblasts or keratinocytes). The patient's cells would be isolated, genetically engineered to contain an additional Factor IX gene that directs production of the missing Factor IX, and reintroduced into the patient. The patient is then capable of producing his own Factor IX and is no longer a hemophiliac.
When “cloned” libraries of genetic material are made for investigative or diagnostic purposes, total DNA is randomly digested with one or more restriction enzymes, and the DNA fragments thus made are ligated to a plasmid vector, which is capable of replicating in a host such as E. coli. The cell maintains several copies of the vector, and this makes it possible to isolate vector DNA that has the various inserted (cloned) fragments. It then becomes possible to analyze the inserted or cloned fragments for their DNA sequence and function. It is important to isolate “transformed” single colonies where each bacterium contains the same fragment and no other for the analysis, and it is also important to recover all the different fragments that have been ligated to the vector as separate colonies.
The efficient recovery of transformants under conditions where only one plasmid molecule and no other enters the transformed bacterial cell is crucial to the making of complete libraries. Plasmids known as derivatives of ColE1 are usually used as vectors, and the bacteria to be transformed are treated with an electroporator to make them permeable to entering DNA. When ColE1 derivatives and an electroporator are used, it has been found that less than 0.1% of the input DNA is recovered, making recovery very inefficient.
E. coli plasmids of the F type were recognized as independent units of replication in the 1950s, and as [physically circular, chromosome-like elements in the 1960s. It was soon discovered that they are maintained approximately at par with the chromosome. Miniplasmids are smaller than the chromosome by a factor of 103. Thus, the time that it takes to synthesize the plasmid DNA with the cellular Pol-III-dependent machine (Kornberg and Baker, 1002) is almost insignificant compared to the time it takes to synthesize the total genome of the cell.
It has been shown that when cells are synchronized for growth, all origins initiate replication synchronously (Boye and Lobner-Olesen, 1990). Replication is a complicated process that is integrated in the cell cycle. The synchronous initiation at all origins indicates that DNA synthesis starts at the same time after all cells have undergone division. This could be the result of synchronous activation of DNA synthesis at all origins, or the result of a sequestration process that prevents initiation during and after duplication of genomes (since genomes duplicate once during each cell cycle), or both. So far the only evidence that has been provided is for a sequestration mechanism (Lu et al., 1994).
Plasmids code for an origin of replication and one or more polypeptides. When a polypeptide has been shown to be absolutely required for plasmid DNA synthesis it has been considered an initiator of replication. The function performed by plasmid “initiators of replication” has thus far not been clarified. The organization of the miniplasmid RepFIC is a member of the IncFII class. Both the region labeled ori (Masai et al., 1983) and the protein RepA have been shown to be essential (Maas et al., 1991).